Skeletal muscle can bear a high load at constant length, or shorten rapidly when the load is low. This force-velocity relationship is the primary determinant of muscle performance in vivo. Here we exploited the quasi-crystalline order of myosin II motors in muscle filaments to determine the molecular basis of this relationship by X-ray interference and mechanical measurements on intact single cells. We found that, during muscle shortening at a wide range of velocities, individual myosin motors maintain a force of about 6 pN while pulling an actin filament through a 6 nm stroke, then quickly detach when the motor reaches a critical conformation. Thus we show that the force-velocity relationship is primarily a result of a reduction in the number of motors attached to actin in each filament in proportion to the filament load. These results explain muscle performance and efficiency in terms of the molecular mechanism of the myosin motor.

The 18ID undulator beamline of the Biophysics Collaborative Access Team at the Advanced Photon Source, Argonne, IL, USA, is a highperformance instrument designed for, and dedicated to, the study of partially ordered and disordered biological materials using the techniques of small-angle X-ray scattering, ®ber diffraction, and X-ray absorption spectroscopy. The beamline and associated instrumentation are described in detail and examples of the representative experimental results are presented.

In aqueous solution some proteins undergo large-scale movements of secondary structures, subunits or domains -referred to as protein 'breathing' -that define a native-state ensemble of structures. These fluctuations are sensitive to the nature and concentration of solutes and other proteins and are thereby expected to be different in the crowded interior of a cell than in dilute solution. Here we use a combination of wide angle x-ray scattering (WAXS) and computational modeling to derive a quantitative measure of the spatial scale of conformational fluctuations in a protein solution. Concentration-dependent changes in the observed scattering intensities are consistent with a model of structural fluctuations in which secondary structures undergo rigid-body motions relative to one another. This motion increases with decreasing protein concentration or increasing temperature. Analysis of a set of five structurally and functionally diverse proteins reveals a diversity of kinetic behaviors. Proteins with multiple disulfide bonds exhibit little or no increase in breathing in dilute solutions. The spatial extent of structural fluctuations appears highly dependent on both protein structure and concentration and is universally suppressed at very high protein concentrations.

The active force of mammalian skeletal muscle is reduced at low temperatures. Caremani et al. reveal that this is due to the rise of a population of myosin motors captured in a refractory state insensitive to muscle activation.

Stretch activation is important in the mechanical properties of vertebrate cardiac muscle and essential to the flight muscles of most insects. Despite decades of investigation, the underlying molecular mechanism of stretch activation is unknown. We investigated the role of recently observed connections between myosin and troponin, called "troponin bridges," by analyzing real-time X-ray diffraction "movies" from sinusoidally stretch-activated Lethocerus muscles. Observed changes in X-ray reflections arising from myosin heads, actin filaments, troponin, and tropomyosin were consistent with the hypothesis that troponin bridges are the key agent of mechanical signal transduction. The time-resolved sequence of molecular changes suggests a mechanism for stretch activation, in which troponin bridges mechanically tug tropomyosin aside to relieve tropomyosin's steric blocking of myosin-actin binding. This enables subsequent force production, with crossbridge targeting further enhanced by stretch-induced lattice compression and thick-filament twisting. Similar linkages may operate in other muscle systems, such as mammalian cardiac muscle, where stretch activation is thought to aid in cardiac ejection.S tretch activation is a striking example of mechanical signal transduction, in which stretching a partially activated muscle yields, after a delay, greater activation. It is observed to varying degrees in all striated muscles, is prominent in vertebrate cardiac muscle where it may underlie the Frank-Starling relationship (1), and is essential to flight in multiple orders of insects, which together comprise ∼75% of insect species (2) and fully half of all animal taxa (3). Since stretch activation was first reported by Pringle (4), a great deal has been learned about how muscular contraction is driven by cyclic myosin-actin interactions (5) which, in vertebrate skeletal muscles, are controlled by calcium's effect on the steric blocking action of troponin-tropomyosin (6). However, even atomic resolution models of myosin-actin (7) and the troponin complex (8, 9) fail to shed any light on the mechanism of activation by stretch. The central question of stretch activation remains: How does mechanical stress convert to myosinactin activation while the requisite [Ca 2þ ] stays constant?Recently we observed cross-bridges between thick (mainly myosin) filaments and thin (mainly actin-troponin-tropomyosin) filaments at the level of the troponin (10). These myosin-troponin connections, referred to here simply as troponin bridges, comprised about 15% of all cross-bridges identified in an insect flight muscle (IFM) quick frozen during an isometric contraction. Troponin bridges represent a newly recognized class of crossbridges, distinct from the "traditional" force-generating myosin cross-bridges that bind to actin target zones halfway between troponins in IFM (10, 11). The direct observation of troponin bridges revived a previously speculative mechanism for stretch activation (12), in which troponin bridges serve as the agent of mechanica...